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Goldilocks and the Three TFs Make Blood Vessels

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Amniotic fluid cells routinely used for prenatal diagnostics can be reprogrammed directly into stable endothelial cells (ECs) that could feasibly repair damaged organs or regenerate blood vessels, researchers report. The reprogramming approach, which bypasses the pluripotent stage, involves that forced expression of three E-twenty six (ETS) family transcription factors, plus transient inhibition of TGFβ to switch on VEGFR2 signaling in the converted cells.

The Weill Cornell Medical College investigators say their studies have shown that AC-derived reprogrammed vascular endothelial cells (rAC-VECs) are essentially locked into a mature endothelial cell state, are highly proliferative and stable, and can generate functional, long-lasting vasculature in experimental mice. Encouragingly, the Weill Cornell team demonstrated that the cells also retain enough plasticity to undergo additional specialization when transplanted into an organ-specific microenvironment: rAC-VECs acquired the morphology of sinusoidal ECs when transplanted into a regenerating liver. Genome-wide transcriptome analyses further confirmed that rAC-VECs express a complete vascular signature that is similar to adult ECs, including human umbilical vein ECs (HUVECs) and adult liver sinusoidal ECs.

And with thousands of amniocenteses being carried out worldwide every year, the team claims it should be possible to collect, cryopreserve, and publicly bank an ethnically diverse range of HLA-typed ACs and rAC-VECs for use in organ regeneration and treating vascular disorders in genetically disparate populations of patients. "Currently, there is no treatment available for a broad range of patients with vascular diseases, including patients who have suffered heart attack, stroke, lung diseases, trauma, emphysema, or even diabetes and neurological disorders," explains Shahin Rafii, M.D., who headed the research, and is the Arthur B. Belfer Professor in Genetic Medicine at Weill Cornell Medical College, and co-director of its Ansary Stem Cell Institute. "Replacing injured or dysfunctional endothelial cells with normal cultured endothelial cells could potentially provide for a novel therapy to treat these diseases that afflict millions of patients worldwide...Selecting the proper immunologically matched endothelial cells for each patient would be akin to blood typing...There are only so many varieties, which are well represented across the amniotic fluid cells that could be obtained, frozen, and banked from wide variety of ethnic groups around the world."

Previous work has already demonstrated that induced vascular endothelial cells (iVECs) can be generated from induced pluriplotent stem cells and human embryonic stem cells (hESCs). However, the stem cell-derived iVECs that don’t proliferate well and have a tendency to "drift" and differentiate into nonvascular lineages, the Weill Cornell investigators state. Similarly, adult cell-derived ECs can’t be readily expanded, and so would be hard to generate at a large scale. And while endothelial progenitor cells and endothelial colony-forming cells do show significant expansion potential when grown in plasma, its not known whether they will maintain their vascular identity after serial passaging.

In their search for a more suitable source of cells for reprogramming into endothelial cells, Dr. Rafii’s team turned to cells found in mid-pregnancy amniotic fluid, which studies had already suggested have the potential to convert into differentiated cell types, if the right triggers could be found. “They are not as plastic and unstable as endothelial cells derived from embryonic cells or as stubborn as those produced from reprogramming differentiated adult cells," explains co-investigator Michael Ginsberg, Ph.D. Rather, he claims, the amniotic cells exhibit exactly the right properties—which the team terms the "Goldilocks Principle"—for producing endothelial cells.

To find the right combination of reprogramming factors needed to trigger this conversion of ACs into endothelial cells, the team looked for transcription factors that are required for vasculogenic specification of ECs and the maintenance of iVECs, using an established model of hESC differentiation into iVECs. They identified a key role for the ETS family transcription factors ETV2, FLI1, and ERG. More specifically, ETV2 was expressed transiently during hESC transition to iVECs, whereas FLI1 and ERG were constitutively expressed.

Building on these findings, the team then carried out a series of experiments to test whether transducing a subset of lineage-committed ACs using the three transcription factors could prompt their conversion into endothelial cells. Their final protocol again involved transient expression of ETV2 (using a doxycycline-dependent inducible (Tet-off) expression system to control ETV2 production), constitutive expression of FLI1 and ERG1, and transient TGFβ suppression. Using this approach the expression of EC genes was switched on in ACs and, importantly, expression of nonvascular genes was switched off. In fact the resulting rAC-EVCs cells were stable and capable of undergoing a 6 x 104-fold expansion in 50 days, while retaining expression of adhesion molecules, extracellular matrix proteins, and angiocrine factors that are necessary for vascular function.

Notably, when matrigel plugs loaded with TGFβ-inhibited rAC-VECs were implanted into experimental mice, the rAV-VECs generated numerous functional vessels that interacted with the host’s vasculature. Similarly, when rAC-VECs were implanted in mice that had gone a partial hepatectomy, about 5–10% of the regenerated liver sinusoidal vessels were made up of the AC-derived vascular endothelial cells. “It was remarkable to see that these cells went right to work building new blood vessels in the liver as well as producing the right growth factors that could potentially regenerate and repair injured organs," Dr. Ginsberg remarks.

Dr Rafii suggests that clinical trials with AC-derived endothelial cells could begin within as little as four years. And continued work should help to identify transcription factors that give ECs a tissue-specific signature, enabling the generation of rAC-VECs that adapt specifically to the physiological and metabolic needs of a particular organ.

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